The CDC estimates that ~300,000 cases of Lyme disease and ~2000 cases of babesiosis occur in the USA every year. Lyme disease is caused by Borrelia burgdorferi spirochetes while the protozoan parasite Babesia microti (referred to as Bm here) is the major causative agent of babesiosis in the USA. Emergence of B. burgdorferi-Bm co-infections in expanding regions of North America and Europe has become a major health concern in the last decade. Synergism or antagonism of these pathogens during co-infections has not yet been described. B. burgdorferi-Bm co-infected patients show more extensive symptoms that persist longer than patients infected with B. burgdorferi alone. Co-infected patients often need hospitalization, and disease in some cases is fatal. Understanding the effects of these pathogens on each other and on the host will ultimately lead to development of better diagnostic, protective and treatment strategies. Limited murine studies conducted until now showed contradictory outcomes of Bm-B. burgdorferi co- infections in different mouse strains. C3H mice are ideal to study the impact of co-infections because this strain exhibits both Lyme disease and babesiosis manifestations similar to humans. Our preliminary data shows that infection with Bm causes anemia, hepatomegaly, and splenomegaly in C3H mice and results in depletion of splenic T and B cells. These changes are associated with a decrease in Bm- and B. burgdorferi-specific antibodies in co-infected mice and both: increased colonization of various tissues and enhanced inflammatory Lyme disease. If Bm infection remains undetected and untreated, such changes in co-infected humans could result in prolonged suffering of patients and could potentially contribute to post- treatment Lyme disease syndrome. We propose to carry out the first extensive study to understand the impact of Bm on B. burgdorferi gene expression, survival and persistence in the susceptible C3H mice and the effect of B. burgdorferi on reducing Bm parasitemia. Based upon our preliminary studies, we hypothesize that: (i) host sex and age are significant biological variables in pathogenesis during Bm-B. burgdorferi co-infection, (ii) depletion of splenic T and B cells by Bm reduces overall antibody production affecting kinetics of B. burgdorferi clearance and increases severity of Lyme disease while stimulation of innate immune response by B. burgdorferi reduces Bm parasitemia, and (iii) modulation of host response by Bm induces specific gene expression in B. burgdorferi to allow long-term survival of spirochetes, tissues colonization, and inflammatory disease. We will: (1) determine the effect of sex and age of mice on Lyme disease and babesiosis during Bm-B. burgdorferi co-infections, (2) determine the effect of sequential B. burgdorferi/Bm infections on Lyme disease, and (3) identify antigenic proteins produced specifically during co-infections that may facilitate long-term B. burgdorferi persistence. A better understanding of co-infections will provide an insight into human disease and identify useful antigenic markers for persistent Lyme disease.